Bipolar switching by HCN voltage sensor underlies hyperpolarization activation

Cowgill, John; Klenchin, Vadim A.; Alvarez-Baron, Claudia P.; Tewari, Debanjan; Blair, Alexander; Chanda, Baron

doi:10.1073/pnas.1816724116

Cited by 33 publications

(40 citation statements)

References 54 publications

(79 reference statements)

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“…However, cutting the S4-S5 linker in non-domain-swapped channels, such as hyperpolarizationactivated HCN channels or depolarization-activated Kv10.1 (EAG) and Kv11.1 (hERG) channels 7,8 , does not prevent gating 16,17 , showing that non-domain-swapped channels do not require a covalent link between S4 and S5. Instead, non-covalent interactions between S4 and S5 have been suggested to be important for hyperpolarization activation of HCN channels 16,[18][19][20] . A recent study suggested that S4 inhibits the PD of HCN channels when S4 is up and that the downward S4 movement at hyperpolarized voltages relieves this inhibition 16 .…”

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confidence: 99%

“…A recent study suggested that S4 inhibits the PD of HCN channels when S4 is up and that the downward S4 movement at hyperpolarized voltages relieves this inhibition 16 . In another study, it was shown that a chimera with the VSD from a hyperpolarization-activated HCN channel and the PD from a depolarization-activated EAG channel could open both in response to depolarizations and hyperpolarizations 20 . The authors, therefore, suggested that S4 functions as a bipolar switch: S4 moves from a common resting position to an inward down position to open hyperpolarization-activated HCN channels, but S4 moves from the common resting position to an outward up position to open depolarization-activated EAG channels.…”

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confidence: 99%

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Gating mechanism of hyperpolarization-activated HCN pacemaker channels

2020

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Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential for rhythmic activity in the heart and brain, and mutations in HCN channels are linked to heart arrhythmia and epilepsy. HCN channels belong to the family of voltage-gated K + (Kv) channels. However, why HCN channels are activated by hyperpolarization whereas Kv channels are activated by depolarization is not clear. Here we reverse the voltage dependence of HCN channels by mutating only two residues located at the interface between the voltage sensor and the pore domain such that the channels now open upon depolarization instead of hyperpolarization. Our data indicate that what determines whether HCN channels open by hyperpolarizations or depolarizations are small differences in the energies of the closed and open states, due to different interactions between the voltage sensor and the pore in the different channels.

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confidence: 99%

Gating mechanism of hyperpolarization-activated HCN pacemaker channels

2020

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“…Recently, it has been proposed that the voltage-dependent gating of HCN and related depolarization-activated KCNH channels is more conserved than previously thought (34). The VSD of HCN channels is capable of activating the channels with depolarization but this ability is masked by rapid inactivation.…”

Section: Discussionmentioning

confidence: 99%

“…However, the S4-S5 linker is not required for the HCN channel opening with hyperpolarization as co-expression of the VSD and pore domain of spHCN (sea urchin HCN) channels as separate proteins generated hyperpolarization activated currents (33). It has been proposed that an extensive interface between the S4 and S5 in HCN channels is responsible for the apparent hyperpolarization-dependent activation in HCN channels (34). Consistent with this hypothesis, coexpression of the PD with the VSD lacking the Cterminal end of the S4 resulted in cNMP modulated but voltage-independent channels (33) and cross-linking the S4 and S5 could lock the channels in the "locked-open" or "locked-closed" states (35,36).…”

Section: Discussionmentioning

confidence: 99%

HCN domain is required for HCN channel expression and couples voltage- and cAMP-dependent gating mechanisms

Wang

Blanco

Hayoz

et al. 2020

Preprint

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Hyperpolarization-activated cyclic nucleotidegated (HCN) channels are major regulators of synaptic plasticity, and rhythmic activity in the heart and brain. Opening of HCN channels requires membrane hyperpolarization and is further facilitated by intracellular cyclic nucleotides (cNMPs).In HCN channels, membrane hyperpolarization is sensed by the membrane-spanning voltage sensor domain (VSD) and the cNMP-dependent gating is mediated by the intracellular cyclic nucleotidebinding domain (CNBD) connected to the poreforming S6 transmembrane domain via the Clinker. Previous functional analysis of HCN channels suggested a direct or allosteric coupling between the voltage-and cNMPdependent activation mechanisms. However, the specifics of the coupling were unclear. The first cryo-EM structure of an HCN1 channel revealed that a novel structural element, dubbed HCN domain (HCND), forms a direct structural link between the VSD and Clinker/CNBD. In this study, we investigated the functional significance of the HCND. Deletion of the HCND prevented surface expression of HCN2 channels. Based on the HCN1 structure analysis, we identified R237 and G239 residues on the S2 of the VSD that form direct interactions with I135 on the HCND. Disrupting these interactions abolished HCN2 currents. We then identified three residues on the C-linker/CNBD (E478, Q382 and H559) that form direct interactions with residues R154 and S158 on the HCND. Disrupting these interactions affected both voltage-and cAMPdependent gating of HCN2 channels. These findings indicate that the HCND is necessary for the surface expression of HCN channels, and provides a functional link between the voltage-and cAMP-dependent mechanisms of HCN channel gating.Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are peculiar members of the voltage-gated potassium channel superfamily. Unlike other potassium channels, which are depolarization-activated and highly selective for K + , HCN channels are activated by membrane hyperpolarization and permeate both Na + and K + (1,2). The opening of HCN channels is further facilitated by cyclic nucleotides (1-3).

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“…Furthermore, the rearrangement is similar to the S4 movement in VSD2 of the depolarization-activated two-pore channel 1 (TPC1) inferred from structures of mouse TPC1 (up state) and Ba 2+ -inhibited Arabidopsis TPC1 (down state) at 0 mV 26,27 (Supplementary Video 6), but is incompatible with other models proposing that the S4 helix is relatively immobile 28 . Moreover, considering the tight packing of S4 and S5 helices in HCN channels 11 , the downward and tilting movement of the S4 helix could bypass the short S4-S5 linker to influence S5 directly, thus allowing the channels to open 29,30 . Our study has revealed, for the first time, the detailed movement of the S4 helix of a VSD during hyperpolarization.…”

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The HCN Channel Voltage Sensor Undergoes A Large Downward Motion During Hyperpolarization

Dai

Aman

DiMaio

et al. 2019

Preprint

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Voltage-gated ion channels (VGICs) underlie almost all electrical signaling in the body 1 . They change their open probability in response to changes in transmembrane voltage, allowing permeant ions to flow across the cell membrane. Ion flow through VGICs underlies numerous physiological processes in excitable cells 1 . In particular, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which operate at the threshold of excitability, are essential for pacemaking activity, resting membrane potential, and synaptic integration 2 . VGICs contain a series of positively-charged residues that are displaced in response to changes in transmembrane voltage, resulting in a conformational change that opens the pore 3-6 . These voltage-sensing charges, which reside in the S4 transmembrane helix of the voltage-sensor domain (VSD) 3 and within the membrane's electric field, are thought to move towards the inside of the cell (downwards) during membrane hyperpolarization 7 . HCN channels are unique among VGICs because their open probability is increased by membrane hyperpolarization rather than depolarization 8-10 . The mechanism underlying this "reverse gating" is still unclear. Moreover, although many X-ray crystal and cryo-EM structures have been solved for the depolarized state of the VSD, including that of HCN channels 11 , no structures have been solved at hyperpolarized voltages. Here we measure the precise movement of the charged S4 helix of an HCN channel using transition metal ion fluorescence resonance energy transfer (tmFRET). We show that the S4 undergoes a significant (~10 Å) downward movement in response to membrane hyperpolarization. Furthermore, by applying constraints determined from tmFRET experiments to Rosetta modeling, we reveal that the carboxyl-terminal part of the S4 helix exhibits an unexpected tilting motion during hyperpolarization activation. These data provide a long-sought glimpse of the hyperpolarized state of a functioning VSD and also a framework for understanding the dynamics of reverse gating in HCN channels. Our methods can be broadly applied to probe short-distance rearrangements in other ion channels and membrane proteins.

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Bipolar switching by HCN voltage sensor underlies hyperpolarization activation

Cited by 33 publications

References 54 publications

Gating mechanism of hyperpolarization-activated HCN pacemaker channels

Gating mechanism of hyperpolarization-activated HCN pacemaker channels

HCN domain is required for HCN channel expression and couples voltage- and cAMP-dependent gating mechanisms

The HCN Channel Voltage Sensor Undergoes A Large Downward Motion During Hyperpolarization

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